There are diverse polysaccharides according to the types of their constituent monosaccharides. The polysaccharides have various molecular weights, properties, and physiological functions and are the popular target of intensive researches.
As is known in the art, coupling of a compound having a functional group capable of condensing with a carboxyl group with that of hyaluronic acid, which is known as a high-molecular weight polysaccharide, decreases the hydrophilicity of the resulting hyaluronic acid derivative and causes the problem of sparse solubility or even insolubility.
One conventionally known technique for coupling carboxyl-linkable, nucleophilic functional group and carboxyl group of hyaluronic acid uses water-soluble carbodiimide (WSC) to allow hyaluronic acid to react with amino group in aqueous solvent (JP 2004-018750 and WO2005/085294). This technique is hereafter referred to as the WSC method. The hyaluronic acid derivative obtained by the WSC method has the problem of sparse solubility or insolubility. According to the description in JP 2004-018750, when the rate of the introduced compound per disaccharide unit of hyaluronic acid (degree of substitution) is not less than 0.05%, the isolated hyaluronic acid derivative may be insoluble in neutral aqueous solvents. When the degree of substitution is not less than 5%, the isolated hyaluronic acid derivative is insoluble in aqueous solvents.
For this insolubility problem, the production method proposed in JP 2004-018750 adds a base to the reaction solution after the condensation reaction of hyaluronic acid. This alkaline treatment allows the compound by the WSC method to be able to dissolve in neutral aqueous solvent. WO2005/085294 also adopts this alkaline treatment step of JP 2004-018750 to prepare a neutral aqueous solution of the resulting hyaluronic acid derivative. Namely, JP 2004-018750 and WO2005/085294 adopt the two-step preparation process including the condensation reaction step by the WSC method and the alkaline treatment step to prepare a hyaluronic acid derivative that is soluble in an aqueous solvent.
As described in JP 2004-018750, the hyaluronic acid derivative prepared by the conventional WSC method is expected to have a change in its higher-order structure, which can be observed as the phenomenon of insolubility in the neutral aqueous solvent. Condensation condition of the WSC method causes the hyaluronic acid derivative to be insoluble in the neutral aqueous solution. The alkaline treatment after the WSC method as described in JP 2004-018750 recovers the solubility of the hyaluronic acid derivative. The recovery of the water solubility is ascribed to a further change or restoration of the higher-order structure which has been changed by the WSC method.
The base-adding step (alkaline treatment) described in JP 2004-018750 further changes or restores the higher-order structure which has been changed in the course of the condensation reaction to regain the once-lost high affinity to the aqueous solvent.
A commercially available condensing agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) works for condensation of a carboxyl group and a carboxyl-linkable, nucleophilic functional group and is mainly used for synthesis of amide compounds by condensation of low-molecular substances. According to Tetrahedron 55 (1999) 13159, DMT-MM activates a carboxyl group in aqueous solvent and helps to form an amide bond with an amino group at a high rate. JP 2005-281372 describes application of DMT-MM for forming an amide bond between a carboxyl group of a carboxyl group-introduced cyclodextrin derivative and an amino group of chitosan. This production method of JP 2005-281372, however, aims to give an insoluble derivative. There has been no report regarding application of DMT-MM for the condensation reaction of a carboxyl group of a high molecular weight polysaccharide (for example, hyaluronic acid) with a carboxyl-linkable, functional group of an organic compound with a view to preparing a water-soluble derivative. There has also been no report describing the properties of an aqueous solution of the resulting derivative or the change of the higher-order structure of the resulting derivative.